The present invention is directed to synthetic Oligomers having phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages and to methods for their synthesis.
Oligomers having naturally occurring phosphodiester internucleosidyl linkages and certain other internucleosidyl linkages do not have chiral centers at the phosphorus atom (or other atom) of the internucleosidyl linkage.
Oligomers having an alkylphosphonate or alkylphosphonothioate internucleosidyl likages have chiral centers at the phosphorus atom of the internucleosidyl linkage. These Oligomers are capable of having either Rp or Sp chirality at each phosphorus, and a particular Oligomer theoretically can have 2.sup.n different diastereomeric forms where n is the number of phosphonate internucleosidyl linkages in the Oligomer. For example, an Oligomer having a total of 10 phosphonate internucleosidyl linkages theoretically has 1,024 diastereomers and an Oligomer having a total of 18 phosphonate internucleosidyl linkages theoretically has 262,144 diastereomers. Binding affinity of the oligomer to its target sequence has been reported to be affected depending on whether a phosphonate or phosphonothioate linkage has the Rp or Sp configuration.
The copending and commonly-assigned U.S. patent application Ser. No. 08/154,013, entitled "Chirally Enriched Synthetic Phosphonate Oligomers", filed Nov. 16, 1993, describes chirally enriched all-alkylphosphonate oligomers which were reported to have enhanced binding affinity for complementary RNA sequences. Synthesis of such chirally enriched oligomers may require special synthetic or purification procedures as compared with oligomers having alkylphosphonate linkages of undefined chirality ("racemic").
In a review article summarizing certain work on antisense agents, disadvantages of poorly hybridizing racemic oligodeoxynucleoside methylphosphonates in cell free extracts were said to be more or less balanced by their proposed advantages*/ in cell culture systems. It was noted that certain reports using a normal (deoxyribonucleoside) octamer with one methylphosphonate linkage found the Oligomer with an R bond to have a melting temperature higher than the Oligomer with an S bond. It was also noted that sequence dependence of methylphosphonate base pairing might be as important as chirality. (Wickstrom, "Antisense DNA Therapeutics Neutral Analogs and Their Stereo-chemistry" in Gene Regulation: Biology of Antisense RNA and DNA, 119 to 132 (Erickson and Izant, eds., Raven Press Ltd., New York (1992)) FNT */ Greater longevity, more efficient cellular uptake and lack of charge.
Kibler-Herzog et al., Anti-Cancer Drug Design, 8: 65-79 (1993) reported studies of melting curves for duplex and triplex formation using 19-mers having all-A or all-T sequences and alternating methylphosphonate phosphodiester, phosphodiester and predominantly methylphosphonate backbones.
Quantin and Wetmur, Biochemistry 28: 1040-1047 (1989) reported studies on the effect of ionic strength on hybridization of oligodeoxynucleotides having methylphosphonate linkages replacing certain numbers of phosphodiester linkages to unmodified oligodeoxynucleotides.
Akhtar et al., Life Sciences 49: 1793-1801 (1991) report studies on antisense DNA oligodeoxynucleotide analogs in certain cellular extracts. An oligonucleotide having alternating methylphosphonate and phosphodiester linkages was reported to have comparable stability, in terms of nuclease degradation, to the corresponding methylphosphonate and phosphorothioate oligonucleotides.
Agrawal et al, Tetrahedron Letters 28 (31): 3539-3542 (1987) reported that an oligodeoxynucleotide having two adjacent methylphosphonate replacing phosphodiester linkages at either end of an oligodeoxynucleotide increased resistance to certain nucleases.